1. Field of the Invention
There is a continuing interest in developing new, simpler and more rapid techniques to detect and measure the presence of an analyte in a sample. The analyte can be any of a wide variety of materials such as drugs, naturally occuring physiological compounds, pollutants, chemicals, contaminants, or the like. In many cases speed is important for the measurement, particularly with certain physiologically active compounds. In other situations, convenience and ease of operation can be major considerations.
In order to quantitate the amount of analyte present in an enzyme immunoassay, the extent of reaction of an enzyme-labelled ligand with antibody must be determined. In the case of heterogeneous enzyme immunoassays, this requires a physical separation of the free and antibody-bound fractions. In order to maximize precision and sensitivity, complete separation of the free and bound fractions should be accomplished with relatively simple and fool proof manipulations. The separation should be accomplished rapidly, preferably without elaborate or expensive equipment. Some configurations employed in enzyme immunoassays include an immobilized antigen, an immobilized antibody, a sandwich method, and/or a bridge or unlabelled antibody.
Exemplary of heterogeneous enzyme immunoassays is the enzyme-linked immunosorbent assay (ELISA). ELISA includes a separation of enzyme-labelled antigen-antibody complex (bound enzyme) from free enzyme-labelled antigen or antibody. The enzymatic activity in the bound or free fraction is quantitated by the enzyme-catalysed conversion of a relatively nonchromatic or nonfluorescent substrate to a highly chromatic or fluorescent product. The various ELISA assay techniques have been classified as either competitive or noncompetitive depending on whether the technique involves a reaction step in which unlabelled antigen and antigen linked to an enzyme or attached to a solid phase compete for a limited number of antibody binding sites, or whether the antigen or antibody to be measured is allowed to react alone with an excess of immune reactant. Whether the approach is competitive or noncompetitive, the ELISA technique involves at least one, and more than likely at least two or three, separation and wash steps. First, there is generally a separation and a wash step after the attachment of antigen or antibody to a solid phase. Secondly, there is a separation and a wash step after incubation of enzyme-labelled antibody. Additionally, in noncompetitive ELISA there is an additional separation and wash step.
In the case of homogenous enzyme immunoassays, no separation of the enzyme labeled antigen-antibody complex from the free enzyme labeled antigen or antibody is required. The enzyme activity of the bond or free fraction can be determined without separation because the enzyme activity changes as a result of binding. Because no separation is employed, these assays frequently are adversely affected by the sample medium and thus only relatively small amounts of sample can be used in the assay medium and the assay sensitivity is thereby reduced. Moreover, it is usually not possible to use excess enzyme labeled reagents because the modulator of the activity of the bound reagent would not be measurable with a large background signal. For these reasons it is sometimes desirable to bind one of the reagents on a solid surface that can be contacted with the sample and/or the enzyme labeled reagent and then separated prior to incubation with a chromogenic substrate.
One convenient and rapid technique that has found wide application is the use of a strip generally comprising of solid rod or sheet on which is attached a pad for conducting an immunochemical reaction. The strip can be dipped in a sample solution containing other reagents and subsequently processed to produce a signal based on the amount of analyte in the original sample. Such an immunochemical strip allows for convenient handling, transfers, separations and the like.
It is desirable to develop a new assay technique which provides for accurate detection of an analyte in a sample and which can be conducted without separation and wash steps and with minimal interference from factors in the sample. It is further desirable that the assay be able to be conducted on relatively small quantities of a sample.
2. Brief Description of the Related Art
Patents disclosing a variety of methods involving separation of bound and unbound antigen include U.S. Pat. Nos. Re. 29,169; 3,949,064; 3,984,533; 3,985,867; 4,020,151; 4,039,652; 4,067,959; 4,108,972; 4,145,406; and 4,168,146. A simultaneous calibration heterogenous immunoassay is disclosed in U.S. Pat. No. 4,533,629. Preferential signal production on a surface in immunoassays is described in U.S. Pat. No. 4,299,916. U.S. Pat. No. 4,391,904 discloses test strip kits in immunoassays and compositions therein.
An apparatus for automatic measurement of the test results of agglutination tests is described in U.S. Pat. No. 4,290,997. An apparatus for microscopic examination of specimens is disclosed in U.S. Pat. No. 4,427,634. A measure and funnel is disclosed in U.S. Pat. No. 3,132,768. A biological fluid sample processing apparatus is disclosed in U.S. Pat. No. 3,545,932. U.S. Pat. No. 2,835,246 describes an apparatus for handling medical specimens. A urine testing apparatus is disclosed in U.S. Pat. No. 3,774,455. U.S. Pat. No. 4,473,530 discloses a compact sanitary urine analysis unit.